Charged-balanced imaging agents

ABSTRACT

The present invention relates to compositions for and methods of optically imaging tissues or cells using imaging agents having desirable in vivo properties that result in improved signal-to-background ratio.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No.R01-CA-115296 awarded by the National Institutes of Health. TheGovernment may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods of optically imaging tissues orcells using imaging agents having desirable in vivo properties thatresult in improved signal-to-background ratio.

BACKGROUND OF THE INVENTION

Near infrared (NIR) fluorescence has potential importance in the medicalfield, particularly in diagnostics and image-guided surgery. However,the availability of suitable fluorophores as imaging agents has been aprimary hindrance. To be clinically viable, the ideal NIR fluorophoreshould have both good optical properties and superior in vivo propertieswith respect to solubility, biodistribution, and clearance. Most currentfluorophores contemplated for use as imaging agents fail in connectionwith their in vivo properties. For example, known fluorophores tend toclear through the liver, which results in undesirable fluorescencethroughout the gastrointestinal tract. And in some cases, knownfluorophores suffer from significant non-specific background uptake innormal tissues, resulting in a low signal-to-background ratio.Accordingly, there is a current need for new and improved NIRfluorescent imaging agents, particularly those that can equilibraterapidly between the intravascular and extravascular spaces and arecleared efficiently by renal filtration. The imaging agents of theinvention are directed toward these and other needs.

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the discovery that if onebalances, or almost balances, the overall charge on an imaging agentmolecule, then the resulting charge-balanced molecule has improved invivo properties that lead to superior clinical imaging characteristics.

In one aspect, the present invention provides methods of imaging tissueor cells, the methods including (a) contacting the tissue or cells withan imaging agent comprising a dye or conjugate thereof, the conjugatecomprising a targeting ligand attached to the dye, wherein the dye orconjugate has a net charge of +1, 0, or −1 and comprises one or moreionic groups; (b) irradiating the tissue or cells at a wavelengthabsorbed by the dye or conjugate; (c) detecting an optical signal fromthe irradiated tissue or cells, wherein the signal-to-background ratioof the detected optical signal is at least about 1.1, thereby imagingthe tissue or cells.

The present invention further provides methods of preparing a dye forimaging tissue or cells, the method including (a) selecting a dye havingpeak absorption at about 500 nm to about 850 nm and peak fluorescentemission at about 550 nm to about 875 nm; (b) optionally modifying thedye to include a linking group; and (c) modifying the dye, andoptionally the linking group, to include one or more ionic groups toachieve a solubility of the dye of at least about 10 μM in 10 mM HEPESsolution at pH 7.4; wherein the one or more ionic groups are selected sothat the net charge of the dye is +1, 0, or −1, and wherein thesignal-to-background ratio of fluorescent emission detected from the dyecompound while imaging is at least about 1.1.

In another aspect, the present invention further provides methods ofpreparing a conjugate for imaging tissue or cells, wherein the conjugateincludes a dye and a targeting ligand. These methods include (a)selecting a dye having peak absorption at about 500 nm to about 850 nmand peak fluorescent emission at about 550 nm to about 875 nm; (b)optionally modifying the dye to include a linking group; (c) modifyingthe dye and optionally the linking group to include one or more ionicgroups to achieve a solubility of at least about 10 μM in 10 mM HEPESsolution at pH 7.4; and (d) conjugating the targeting ligand to the dyeoptionally through the linking group to form the conjugate, wherein thetargeting ligand and the one or more ionic groups are selected so thatthe net charge of the conjugate is +1, 0, or −1, and wherein thesignal-to-background ratio of fluorescent emission detected from theconjugate while imaging is at least about 1.1.

In addition, the present invention includes imaging agents for imagingtissue or cells, wherein the imaging agents include a conjugate which ischaracterized as having detectable fluorescence with asignal-to-background ratio of at least about 1.1, and wherein theconjugate has Formula VI:

wherein constituent variables are defined herein.

The present invention further provides a dye comprising a molecule orion of Formula VIII:

wherein constituent variables are defined herein.

The charge-balanced imaging agents of the invention are particularlyadvantageous because their behavior in vivo is believed to contribute tosuperior optical imaging properties. More specifically, thecharge-balancing is believed to impart good biodistribution andclearance properties to the agents, and reduce undesirable non-specificbinding. These in vivo properties help improve the signal-to-backgroundratio of imaged tissues, leading to higher resolution imaging.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a representation of dye molecules having a range of net charges.

FIG. 2 is a schematic of the synthesis of dye molecule ZW+1 (MM-19).

FIG. 3 is a schematic of the synthesis of dye molecule ZW+5.

FIG. 4 is a representation of five dye molecules, the properties ofwhich were compared in vitro and in vivo.

FIG. 5 is a representation of experimental results relating to in vivobiodistribution and clearance of the dye molecules of FIG. 4 in a ratmodel.

FIG. 6 is a representation of experimental results relating to in vivobiodistribution and clearance of the dye molecules of FIG. 4 in a pigmodel.

FIG. 7 is a representation of two different dyes conjugated to thetargeting ligand cRGD.

FIG. 8 is a representation of in vivo experimental results in rat tumormodels for the two conjugates of FIG. 7.

FIG. 9 is a representation of the preparation of an exampleadamantane-based targeting ligand containing an ionic group forcharge-balancing and moieties that selectively bind to PSMA.

DETAILED DESCRIPTION

The present disclosure relates, inter alia, to imaging agents that arecomposed of a dye molecule optionally conjugated to a targeting ligandthrough a linking group. The imaging agents described herein are usefulin, for example, the detection of abnormal or diseased biologicaltissues and cells. The conjugates are particularly useful for imagingwhole organisms, because they have improved in vivo behavior, such aslow non-specific binding to non-targeted tissues, resulting in animproved signal-to-background ratio in connection with the detectedoptical signal. It is believed that these improved in vivo propertiesresult from the balancing of formal charges on the conjugate, renderinga “charge-balanced” molecule having a net charge that is neutral orclose to neutral.

Imaging Methods

The new methods of imaging tissue or cells include the following basicsteps:

(a) contacting the tissue or cells with an imaging agent comprising adye or conjugate thereof, the conjugate comprising a targeting ligandattached to the dye, wherein the dye or conjugate has a net charge of+1, 0, or −1 and comprises one or more ionic groups;

(b) irradiating the tissue or cells at a wavelength absorbed by the dyeor conjugate;

(c) detecting an optical signal from the irradiated tissue or cells,

wherein the signal-to-background ratio of the detected optical signal isat least about 1.1, thereby imaging the tissue or cells.

The imaging agents described herein are substances or molecules that canbe used to image tissues or cells, such as those of a living organism,for purposes of diagnosis, therapy, image-guided surgery, and the like.In some embodiments, the organism is a mammal, such as a human.

The imaging agents generally contain a dye that is capable of absorptionof electromagnetic radiation, typically in the ultraviolet (UV),visible, or near infrared (NIR) range. The imaging agent can also becapable fluorescent emission, such as in the visible or NIR range. Theoptical signal detected from the dye or conjugate can be, for example,absorption or fluorescent emission. In some embodiments, fluorescentemission from the dye is the primary optical signal detected for imagingpurposes. In some embodiments, the dye has a peak absorbance at about525 nm to about 850 nm, at about 550 nm to about 825 nm, about 600 nm toabout 825 nm, about 700 nm to about 825 nm, or at about 750 nm to about825 nm. In some embodiments, the dye has a peak fluorescent emission atabout 700 nm to 875 nm, about 725 to about 850 nm, about 750 to about850 nm, or about 775 to about 850 nm.

Suitable dyes for imaging by fluorescent emission include the class ofcyanine dyes which are cationic molecules where two cyclic groups arelinked through a methine or polymethine bridge. See the followingreferences for examples of various cyanine dye derivatives: Mojzych, M.et al. “Synthesis of Cyanine Dyes” Top. Heterocycl. Chem. (2008) 14:1-9;Sysmex Journal International (1999), Vol. 9, No. 2, pg 185 (appendix);Strekowski, L. et al. Synthetic Communications (1992), 22(17),2593-2598; Strekowski, L. et al. J. Org. Chem. (1992) 57, 4578-4580;Narayanan, N. et al. J. Org. Chem. (1995), 60(8), 2391-2395; Makin, S.M. et al. Journal of Organic Chemistry of the USSR (1977) 13(6), part 1,1093-1096; Lee, H. et al. J. Org. Chem. (2006) 71, 7862-7865, WO2009/006443, WO 2008/015415, WO 2007/136996, WO 2007/005222, WO2003/082988, WO 2001/090253, U.S. Ser. No. 12/376,243 (filed Feb. 3,2009), and U.S. Ser. No. 12/376,225 (filed Feb. 3, 2009), each of whichis incorporated herein by reference in its entirety. Example dyes andtheir conjugates suitable for use in the present imaging methods aredescribed herein.

The imaging agents can include conjugates, which refers to a dye whichis conjugated to a targeting ligand. The “targeting ligand” is a moietythat binds with some specificity or selectivity to a particular tissueor biological target. The tissue or biological target can include normaltissues as well as abnormal or diseased tissues. Targeting ligands canbe selected from specific proteins, protein fragments, peptides,antibodies, carbohydrates, or antigens described, e.g., in Frangioni etal. in “Modified PSMA Ligands and Uses Related Thereto,” WO 02/098885,filed on Feb. 7, 2002 (now issued as U.S. Pat. No. 6,875,886).

An example targeting ligand is the cRGD peptide, which selectively bindsto the biological target α_(V)β₃ integrin. It is known that thisintegrin is overexpressed by various tumors, and thus, these RGDtargeting peptides enable the dyes to preferentially label tumors thatoverexpress these integrins. Other targeting ligands include melanocytestimulating hormone (MSH), which binds to melanoma cells; or bombesin,somatostatin, or Sandostatin™ (synthetic), which target somatostatinreceptors. Other targeting ligands include “KUE” and other smallmolecules, which selectively bind to the biological targetprostate-specific membrane antigen (PSMA) (See, Humblet, V. et al. Mol.Imaging, 2005, 4: 448-62; Misra P. et al. J. Nucl. Med. 2007, 48:1379-89; Chen, Y., et al. J. Med. Chem, 2008, 51: 7933-43; Chandran, S.S., et al. Cancer Biol. Ther., 2008, 7:974-82; Banerjee, S. R., J. Med.Chem. 2008, 51: 4504-17; Mease, R. C., et al. Clin. Cancer Res., 2008,14:3036-43; Foss, C. A. et al. Clin. Cancer. Res., 2005, 11:4022-8, eachof which is incorporated herein by reference in its entirety). Examplesof suitable targeting ligands are described elsewhere herein.

The imaging agents are generally “charge-balanced,” unless otherwisespecified, which refers to having a net overall charge of zero, or closeto zero, such as or −1. Charge-balancing occurs when negatively chargedsubstituents on the imaging agent are countered by the presence of anequal number, or close to an equal number, of positively chargedsubstituents on the same molecule, and vice versa. In some embodiment,the net charge is 0 or +1. In some embodiments, the net charge is 0. Insome embodiments, the net charge is +1. In further embodiments, the netcharge is −1. The value “n” in the formulae provided herein representsnet charge.

The imaging agents described herein generally have improved“signal-to-background ratio” (SBR) compared to presently knownfluorescent imaging agents. The improvement in SBR is believed to be aresult of improved in vivo properties due to “charge-balancing.” SBR isa measure of the intensity of the fluorescent signal obtained from atarget (peak signal) over the measure of the intensity of thefluorescent signal obtained nearby the target (background signal), thetarget being the tissues or cells targeted by the imaging agent. SBRmeasurements can be readily obtained through routine measurementprocedures. For fluorescent imaging systems, and other optical-typesystems, digital images recording optical signals of the targetfacilitate SBR measurement. Higher SBR values are more desirable,resulting in greater resolution of the imaged tissues. In someembodiments, the imaging agents achieve an SBR of at least about 1.1(i.e., peak signal is at least 10% over background). In furtherembodiments, the imaging agents achieve an SBR of at least about 1.2, atleast about 1.3, at least about 1.4, at least about 1.5, at least about1.6, at least about 1.7, at least about 1.8, at least about 1.9, or atleast about 2.0. In yet further embodiments, the imaging agents achievean SBR of about 1.1 to about 50, about 1.5 to about 30, about 2.0 toabout 20, about 2.0 to about 5.0, or about 5.0 to about 10.

The imaging agents generally include one or more ionic groups. In someembodiments, the imaging agents include two or more, three or more, fouror more, or five or more ionic groups. Ionic groups serve to increasesolubility of the generally hydrophobic dye portions of the imagingagent, thus, improving biodistribution. The ionic groups can be locatedon any portion of the imaging agent, such as the dye portion, thetargeting ligand, or both.

The term “ionic group” refers to a moiety comprising one or more chargedsubstituents. The “charged substituent” is a functional group that isgenerally anionic or cationic in substantially neutral aqueousconditions (e.g. a pH of about 6.5 to 8.0, or preferably aboutphysiological pH (7.4)). Examples of charged anionic substituentsinclude anions of inorganic and organic acids such as sulfonate (—SO₃¹⁻), supinate, carboxylate, phosphinate, phosphonate, phosphate, andesters (such as alkyl esters) thereof. In some embodiments, the chargedsubstituent is sulfonate. Examples of charged cationic substituentsinclude quaternary amines (—NR₃ ⁺), where R is independently selectedfrom C₁₋₆ alkyl, aryl, and arylalkyl. Other charged cationicsubstituents include protonated primary, secondary, and tertiary amines,and well as guanidinium. In some embodiments, the charged substituent is—N(CH₃)₃ ⁺. Further examples of ionic groups are described infra.

The imaging agents described herein generally have good solubility insubstantially neutral aqueous media, and in particular, blood and bloodserum. In some embodiments, the imaging agent has a solubility in 10 mMHEPES solution, pH 7.4, of at least about 10 μM. In further embodiments,the imaging agent has a solubility in 10 mM HEPES solution, pH 7.4, ofat least about 15 μM, at least about 20 μM, at least about 25 μM, atleast about 30 μM, at least about 40 μM, or at least about 50 μM.

The imaging agents can be neutral molecules or salts. For example, ifthe dye or dye conjugate is charged, the imaging agent can be or containa salt or acid (or combination thereof) of the dye or dye conjugate. Forpositively charged dyes or conjugates, suitable counter ions includeanions such as fluoride, chloride, bromide, iodide, acetate,perchlorate, PF₆ ⁻, and the like. For negatively charged dyes orconjugates, suitable counterions include cations such as Na⁺, K⁺, andquaternary amines.

The imaging agents of the invention can be administered by any suitabletechnique, including both enteral and parenteral methods. In someembodiments, the imaging agents can be formulated into pharmaceuticallyacceptable formulations and administered intravenously to an organismfor imaging. The dosed organism can be imaged using, for example, aFLARE™ Image-Guided Surgery System, which is a continuous-wave (CW)intraoperative imaging system that is capable of simultaneous, real-timeacquisition and display of color video (i.e., surgical anatomy) and twochannels of invisible NIR fluorescent (700 nm and 800 nm) light. Theimaging system can irradiate the dosed organism with radiation absorbedby the imaging agent, and detect optical signals, such as NIRfluorescence, eminating from the targeted portions of the organismcontaining the imaging agent. The detected signals can be recorded andanalyzed by obtaining digital images or video of the subject organism,thereby facilitating diagnostic procedures and image-guided medicaltechniques.

Dyes and Conjugates

In some embodiments, the dyes or conjugates of the invention can haveFormula I:

wherein:

n is +1, 0, or −1;

W is a C₁ methine or a C₂₋₁₃ polymethine group optionally substitutedwith 1, 2, 3, or 4 substituents independently selected from C₁₋₆alkyl, areactive linking group, or a moiety comprising a linking group and atargeting ligand, wherein two substituents, together with the atoms towhich they are attached and optionally one or more methine groups of W,optionally form a 5-, 6-, or 7-membered carbocycle or a 5-, 6-, or7-membered heterocycle, wherein said carbocycle or heterocycle isoptionally substituted with 1, 2, 3, 4, 5, or 6 substituentsindependently selected from an ionic group, a non-ionic oligomeric orpolymeric solubilizing group, halo, C₁₋₆ alkyl, aryl, and heteroaryl;

Ring A and Ring B are independently selected from a monocyclicheterocycle, a bicyclic heterocycle, a tricyclic heterocycle, amonocyclic a carbocycle, a bicyclic carbocycle, and a tricyliccarbocycle, wherein one of Rings A and B is optionally charged;

R^(A) and R^(B) are independently selected from H, an ionic group, anon-ionic oligomeric or polymeric solubilizing group, halo, C₁₋₆ alkyl,aryl, and heteroaryl, wherein said alkyl, aryl, and heteroaryl groupsare optionally substituted with 1, 2, 3, 4, or 5 groups independentlyselected from halo, cyano, nitro, and C₁₋₄ haloalkyl;

x and y are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, and 11; wherein at least one R^(A) and R^(B) is present and is anionic group.

In some embodiments, at least two of R^(A) and R^(B) are present and areionic groups. In further embodiments, at least three of R^(A) and R^(B)are present and are ionic groups. In yet further embodiments, at leastfour of R^(A) and R^(B) are present and are ionic groups. In someembodiments, at least one ionic group is a cationic group. In furtherembodiments, at least two ionic groups are cationic groups.

In some embodiments, Ring A is selected from a monocyclic heterocycle, abicyclic heterocycle, and a tricyclic heterocycle.

In some embodiments, Ring A is selected from a bicyclic heterocycle anda tricyclic heterocycle, wherein Ring A has a formal charge of +1.

In some embodiments, Ring A has the Formula:

wherein:

Ring A is a bicyclic or tricyclic heterocycle;

R^(A) is H, an ionic group, a non-ionic oligomeric or polymericsolubilizing group, halo, C₁₋₆ alkyl, aryl, and heteroaryl, wherein saidalkyl, aryl, and heteroaryl groups are optionally substituted with 1, 2,3, 4, or 5 groups independently selected from halo, cyano, nitro, andC₁₋₄ haloalkyl;

R^(A′) is an ionic group, a non-ionic oligomeric or polymericsolubilizing group, C₁₋₆ alkyl, aryl, and heteroaryl, wherein saidalkyl, aryl, and heteroaryl groups are optionally substituted with 1, 2,3, 4, or 5 groups independently selected from halo, cyano, nitro, andC₁₋₄ haloalkyl; and

t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, the dye or conjugate has Formula II:

wherein:

n is 0, or −1;

W is a C₁ methine or a C₂₋₁₃ polymethine group optionally substitutedwith 1, 2, 3, or 4 substituents independently selected from C₁₋₆ alkyl,a reactive linking group, or a moiety comprising a linking group and atargeting ligand, wherein two substituents, together with the atoms towhich they are attached and optionally one or more methine groups of W,optionally form a 5-, 6-, or 7-membered carbocycle or a 5-, 6-, or7-membered heterocycle, wherein said carbocycle or heterocycle isoptionally substituted with 1, 2, 3, 4, 5, or 6 substituentsindependently selected from an ionic group, a non-ionic oligomeric orpolymeric solubilizing group, halo, C₁₋₆ alkyl, aryl, and heteroaryl;

Ring A and Ring B are independently selected from monocyclicheterocycle, bicyclic heterocycle, and tricyclic heterocycle;

R^(A) and R^(B) are independently selected from H, an ionic group, anon-ionic oligomeric or polymeric solubilizing group, halo, C₁₋₆ alkyl,aryl, and heteroaryl, wherein said alkyl, aryl, and heteroaryl groupsare optionally substituted with 1, 2, 3, 4, or 5 groups independentlyselected from halo, cyano, nitro, and C₁₋₄ haloalkyl;

R^(A′) and R^(B′) are independently selected from an ionic group, anon-ionic oligomeric or polymeric solubilizing group, C₁₋₆ alkyl, aryl,and heteroaryl, wherein said alkyl, aryl, and heteroaryl groups areoptionally substituted with 1, 2, 3, 4, or 5 groups independentlyselected from halo, cyano, nitro, and C₁₋₄ haloalkyl; and

u and t are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,and 10;

wherein at least one R^(A), R^(B), R^(A′), R^(B′), and R^(W) is presentand is an ionic group, and any other substituents are as defined infraand supra.

In some embodiments, at least two R^(A), R^(B), R^(A′), R^(B′), andR^(W) are present and are ionic groups. In further embodiments, at leastthree R^(A), R^(B), R^(A′), R^(B′), and R^(W) are present and are ionicgroups. In yet further embodiments, at least four R^(A), R^(B), R^(A′),R^(B′), and R^(W) are present and are ionic groups. In some embodiments,at least one R^(A), R^(B), R^(A′), R^(B′), and R^(W) is a cationicgroup. In some embodiments, at least two R^(A), R^(B), R^(A′), R^(B′),and R^(W) are cationic groups.

In some embodiments, the dye or conjugate has Formula III:

wherein:

G is independently selected from H, C₁₋₆ alkyl, a moiety comprising alinking group, and a moiety comprising a targeting ligand;

Y is O, S, CR¹¹R¹², NR^(n), —CR¹¹═CR¹²—, or —CR¹¹═N—;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are independently selected from H, anionic group, a non-ionic oligomeric or polymeric solubilizing group,halo, C₁₋₆ alkyl, aryl, and heteroaryl, wherein said alkyl, aryl, andheteroaryl groups are optionally substituted with 1, 2, 3, 4, or 5groups independently selected from halo, cyano, nitro, and C₁₋₄haloalkyl;

or two adjacent R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ groups, together withthe atoms to which they are attached, form a fused 5-7 membered aryl,heteroaryl, cycloalkyl, or heterocycloalkyl group, each optionallysubstituted with 1, 2, 3, 4, or 5 substituents independently selectedfrom halo, cyano, nitro, and C₁₋₄ haloalkyl;

R⁹, R¹⁰, and R^(n) are independently selected from an ionic group, anon-ionic oligomeric or polymeric solubilizing group, C₁₋₆ alkyl, aryl,and heteroaryl, wherein said alkyl, aryl, and heteroaryl groups areoptionally substituted with 1, 2, 3, 4, or 5 groups independentlyselected from halo, cyano, nitro, and C₁₋₄ haloalkyl;

R¹¹ and R¹² are independently selected from C₁₋₄ alkyl optionallysubstituted with 1, 2, or 3 halo;

R¹⁵ and R¹⁶ are independently selected from H and C₁₋₆ alkyl;

-   -   or R¹⁵ and R¹⁶, together with the atoms to which they are        attached and optionally one or more —CH═ moieties, form a        6-membered aryl or cycloalkyl group, each optionally substituted        with 1, 2, 3, 4, 5, or 6 substituents independently selected        from an ionic group, a non-ionic oligomeric or polymeric        solubilizing group, halo, C₁₋₆ alkyl, aryl, and heteroaryl; and

v is 0, 1, 2, 3, 4, or 5,

wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ isan ionic group, and any other substituents are as defined infra andsupra.

In some embodiments, no more than one G is a moiety comprising a linkinggroup or a moiety comprising a targeting ligand. In some embodiments,least two of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ areionicgroups. In further embodiments, least three of R¹, R², R³, R⁴, R⁵,R⁶, R⁷, R⁸, R⁹, and R¹⁰ are ionic groups. In yet further embodiments,least four of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are ionicgroups. In some embodiments, at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, and R¹⁰ is a cationic group. In further embodiments, at leasttwo of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are cationic groups.

In some embodiments, the dye or conjugate has Formula IV:

wherein R¹³ and R¹⁴ are independently selected from C₁₋₄ alkyloptionally substituted with 1, 2, or 3 halo, and any other substituentsare as defined infra and supra

In some embodiments, no more than one G is a moiety comprising a linkinggroup or a moiety comprising a targeting ligand. In some embodiments,least two of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are ionicgroups. In further embodiments, least three of R¹, R², R³, R⁴, R⁵, R⁶,R⁷, R⁸, R⁹, and R¹⁰ are ionic groups. In yet further embodiments, leastfour of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are ionic groups. Insome embodiments, at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,and R¹⁰ is a cationic group. In further embodiments, at least two of R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are cationic groups.

In some embodiments, the dye or conjugate has Formula V:

wherein R²², R²³, R²⁴, R²⁵, R²⁶, and R²⁷ are independently selected froman ionic group, a non-ionic oligomeric or polymeric solubilizing group,halo, C₁₋₆ alkyl, aryl, and heteroaryl, and any other substituents areas defined infra and supra.

In some embodiments, no more than one G is a moiety comprising a linkinggroup or a moiety comprising a targeting ligand. In some embodiments,least two of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are ionicgroups. In further embodiments, least three of R¹, R², R³, R⁴, R⁵, R⁶,R⁷, R⁸, R⁹, and R¹⁰ are ionic groups. In yet further embodiments, leastfour of R′, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are ionic groups. Insome embodiments, at least one of R′, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,and R¹⁰ is a cationic group. In further embodiments, at least two of R¹,R², R³, R4, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are cationic groups.

Conjugates

Suitable conjugates described herein can be characterized by Formula VI:

wherein:

TL is a targeting ligand comprising at least one binding moiety thatbinds to a biological target;

L is a linking moiety;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are independently selected from H, anionic group, a non-ionic oligomeric or polymeric solubilizing group,halo, C₁₋₆ alkyl, aryl, and heteroaryl, wherein said alkyl, aryl, andheteroaryl groups are optionally substituted with 1, 2, 3, 4, or 5groups independently selected from halo, cyano, nitro, and C₁₋₄haloalkyl; or two adjacent R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ groups,together with the atoms to which they are attached, form a fused 5-7membered aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group, eachoptionally substituted with 1, 2, 3, 4, or 5 substituents independentlyselected from halo, cyano, nitro, and C₁₋₄ haloalkyl;

R⁹ and R¹⁰ are independently selected from an ionic group, a non-ionicoligomeric or polymeric solubilizing group, C₁₋₆ alkyl, aryl, andheteroaryl, wherein said alkyl, aryl, and heteroaryl groups areoptionally substituted with 1, 2, 3, 4, or 5 groups independentlyselected from halo, cyano, nitro, and C₁₋₄ haloalkyl;

R¹¹, R¹², R¹³, and R¹⁴ are independently selected from C₁₋₄ alkyloptionally substituted with 1, 2, or 3 halo;

R¹⁵ and R¹⁶ are independently selected from H and C₁₋₆ alkyl;

or R¹⁵ and R¹⁶ together with the —CH═CH—CH═ moiety which they span forma 6-membered aryl or cycloalkyl group, each optionally substituted with1, 2, 3, or 4 substituents independently selected from an ionic group, anon-ionic oligomeric or polymeric solubilizing group, halo, C₁₋₆ alkyl,aryl, and heteroaryl; and

n is +1, 0, or −1.

In some embodiments, at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,and R¹⁰ is an ionic group. In further embodiments, at least two of R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are ionic groups. In yet furtherembodiments, at least three of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, andR¹⁰ are ionic groups. In yet further embodiments, at least four of R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are ionic groups.

In some embodiments, n is 0. In further embodiments, n is +1. In yetfurther embodiments, n is −1.

In some embodiments, the ionic group is independently selected from:

(a) a charged substituent; and

(b) a C₁₋₂₀ alkyl group substituted with one or more chargedsubstituents, and optionally substituted with 1, 2, 3, 4, 5, or 6substituents independently selected from halo, cyano, nitro, and C₁₋₄haloalkyl,

wherein 0, 1, 2, 3, 4, 5, or 6 carbon atoms of the alkyl group areindividually replaced with O, S, C(O), C(O)O, NR′, C(O)NR′, SO, SO₂,SO₂NR′, wherein R′ is H or C₁₋₆ alkyl, with the proviso that thereplacement does not result in an unstable moiety.

In some embodiments, the charged substituent is selected from sulfonateor a quaternary amine of formula —NR₃ ⁺, wherein R is independentlyselected from C₁₋₆ alkyl, aryl, and arylalkyl.

In some embodiments, the C₁₋₂₀ alkyl group substituted with one or morecharged substituents has the Formula:

wherein y and z are independently selected from 1, 2, 3, 4, 5, 6, 7, and8.

In some embodiments, the C₁₋₂₀ alkyl group substituted with one or morecharged substituents is independently selected from:

In some embodiments, the C₁₋₂₀ alkyl group substituted with one or morecharged substituents is a Zwitterionic group. For example, theZwitterionic group can comprise a sulfonate group and a quaternary amineof formula —NR₃ ⁺, wherein R is independently selected from C₁₋₆ alkyl,aryl, and arylalkyl.

In some embodiments, R¹¹, R¹², R¹³, and R¹⁴ are each methyl.

In some embodiments, R¹⁵ and R¹⁶ together with the —C═C—C— moiety whichthey span form a 6-membered aryl or cycloalkyl group, each optionallysubstituted with 1, 2, 3, or 4 substituents independently selected froman ionic group, a non-ionic oligomeric or polymeric solubilizing group,halo, C₁₋₆ alkyl, aryl, and heteroaryl.

In some embodiments, R¹⁵ and R¹⁶ together form —CH₂—CH₂—CH₂—.

In some embodiments, L has the formula:

wherein:

E is absent, O or S;

Q is (CH₂)_(q) or a non-ionic oligomeric or polymeric solubilizingmoiety;

J is C(O), C(O)O, or C(O)NH;

R¹⁷, R¹⁸, R¹⁹, and R²⁰ are independently selected from H, an ionicgroup, a non-ionic oligomeric or polymeric solubilizing moiety, halo,cyano, nitro, C₁₋₄ alkyl, C₁₋₄ alkoxy, and C₁₋₄ haloalkyl; and

q is 0, 1, 2, 3, 4, 5, 6, 7, or 8,

In some embodiments, the conjugate has Formula VII:

wherein the substituents are defined supra and infra, and wherein atleast one of R², R⁶, R⁹, and R¹⁰ is an ionic group. In some embodiments,n is +1. In further embodiments, two of R², R⁶, R⁹, and R¹⁰ are cationicgroups. In yet further embodiments, two of R², R⁶, R⁹, and R¹⁰ arecationic groups and two of R², R⁶, R⁹, and R¹⁰ are anionic groups.

In some embodiments, the conjugate has the Formula VIII:

wherein:

n is 0, +1, or −1; and

TL is a targeting ligand.

Dyes

Suitable dyes described herein include a molecule or ion of FormulaVIII:

wherein:

L′ is a reactive linking group;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are independently selected from H, anionic group, a non-ionic oligomeric or polymeric solubilizing group,halo, C₁₋₆ alkyl, aryl, and heteroaryl, wherein said alkyl, aryl, andheteroaryl groups are optionally substituted with 1, 2, 3, 4, or 5groups independently selected from halo, cyano, nitro, and C₁₋₄haloalkyl;

or two adjacent R¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ groups, together withthe atoms to which they are attached, form a fused 5-7 membered aryl,heteroaryl, cycloalkyl, or heterocyclocalkyl group, each optionallysubstituted with 1, 2, 3, 4, or 5 substituents independently selectedfrom halo, cyano, nitro, and C₁₋₄ haloalkyl;

R⁹ and R¹⁰ are independently selected from an ionic group, a non-ionicoligomeric or polymeric solubilizing group, C₁₋₆ alkyl, aryl, andheteroaryl, wherein said alkyl, aryl, and heteroaryl groups areoptionally substituted with 1, 2, 3, 4, or 5 groups independentlyselected from halo, cyano, nitro, and C₁₋₄ haloalkyl;

R¹¹, R¹², R¹³, and R¹⁴ are independently selected from C₁₋₄ alkyloptionally substituted with 1, 2, or 3 halo;

R¹⁵ and R¹⁶ are independently selected from H and C₁₋₆ alkyl;

or R¹⁵ and R¹⁶ together with the —C═C—C— moiety which they span form a6-membered aryl or cycloalkyl group, each optionally substituted with 1,2, 3, or 4 substituents independently selected from an ionic group, anon-ionic oligomeric or polymeric solubilizing group, halo, C₁₋₆ alkyl,aryl, and heteroaryl; and

p is −6, −5, −4, −3, −2, −1, 0, +1, +2, +3, +4, +5, or +6.

In some embodiments, at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,and R¹⁰ is an ionic group comprising at least one cationic substituent.

In some embodiments, L′ comprises a —COOH group or a —C(O)O—NHS group.

In some embodiments, L′ has the formula:

wherein:

E is absent, O or S;

Q is (CH₂)_(q) or a non-ionic oligomeric or polymeric solubilizingmoiety;

R¹⁷, R¹⁸, R¹⁹, and R²⁰ are independently selected from H, an ionicgroup, a non-ionic oligomeric or polymeric solubilizing moiety, halo,cyano, nitro, C₁₋₄ alkyl, C₁₋₄ alkoxy, and C₁₋₄ haloalkyl;

R²¹ is H or N-succinimidyl; and

-   -   q is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

In some embodiments, the dye comprises a molecule or ion of Formula IX:

wherein the substituents are defined supra and infra, and wherein atleast one of R², R⁶, R⁹, and R¹⁰ is an ionic group comprising at leastone cationic substituent.

In some embodiments, p is 0 or +1. In further embodiments, p is +1.

In some embodiments, at least two of R², R⁶, R⁹, and R¹⁰ are ionicgroups each comprising at least one cationic substituent.

In some embodiments, the dye compound has Formula X:

DEFINITIONS AND ADDITIONAL EMBODIMENTS

As used herein, “targeting ligand” (TL) refers to any molecular entitythat contains a binding moiety that binds with some specificity orselectivity to a biological target, and is the primary means for theconjugates of the invention to bind to specific tissues in an organismor sample. Targeting ligands can further include charged functionalgroups that would balance charge on a conjugated dye molecule.Generally, it is desired that the charge on the targeting ligandsubstantially neutralizes any charge on the dye compound such that thetotal net charge of the conjugate is −1, 0, or +1. In some embodiments,the total net charge is 0.

The targeting ligand can be covalently attached to the reactive linkinggroup of a dye compound of the invention through standard couplingprocedures. For example, the carboxyl or activated carboxyl group of thereactive linking group can react with a nucleophilic functionality onthe targeting ligand, such as an amine or alkoxy derivative, to form anamide or ester linkage. Additional details for the conjugation of dyescan be found in WO 2008/017074 and in Frangioni et al. MolecularImaging, Vol. 1(4), 354-364 (2002), each of which is incorporated hereinby reference in its entirety.

The targeting ligand can further include a molecular scaffold moiety towhich the binding moiety and other groups can attach. For example, themolecule scaffold can bear one or more of the following: (1) a moietydesigned to react with the reactive linking group of the dye to form acovalent bond, (2) a charge balancing moiety, such as any of the ionicgroups described herein, and (3) a moiety that binds to the biologicaltarget. An example of a molecular scaffold is an adamantane derivative,such as described in U.S. Pat. App. Pub. No. 2006/0063834, which isincorporated herein by reference in its entirety, and illustrates thepreparation of a targeting ligand that incorporates an adamantanescaffold. Specifically, the adamantane core holds (1) an amino groupcapable of reacting with the dye compounds, (2) a charge-balancingmoiety that will neutralize a negative charge on the dye molecule, and(3) two moieties that bind to the biological target PSMA. For adescription moieties that bind to PSMA, see, Humblet, V. et al. Mol.Imaging, 2005, 4: 448-62; Misra P. et al. J. Nucl. Med. 2007, 48:1379-89; Chen, Y., et al. J. Med. Chem, 2008, 51: 7933-43; Chandran, S.S., et al. Cancer Biol. Ther., 2008, 7:974-82; Banedee, S. R., J. Med.Chem. 2008, 51: 4504-17; Mease, R. C., et al. Clin. Cancer Res., 2008,14:3036-43; Foss, C. A. et al. Cancer. Res., 2005, 11:4022-8, each ofwhich is incorporated herein by reference in its entirety.

As used herein, the teen “contacting” refers to the bringing together ofsubstances in physical contact such that the substances can interactwith each other. For example, when an imaging agent is “contacted” withtissue or cells, the tissue or cells can interact with the imagingagent, for example, allowing the possibility of binding interactionsbetween the agent and molecular components of the tissue or cells.“Contacting” is meant to include the administration of a substance suchas an imaging agent of the invention to an organism. Administration canbe, for example, oral or parenteral.

As used herein, the term “ionic group” refers to a moiety comprising oneor more charged substituents. The “charged substituent” is a functionalgroup that is generally anionic or cationic when in substantiallyneutral aqueous conditions (e.g. a pH of about 6.5 to 8.0 or aboutphysiological pH (7.4)). As recited above, examples of charged anionicsubstituents include anions of inorganic and organic acids such assulfonate (—SO₃ ¹⁻), sulfinate, carboxylate, phosphinate, phosphonate,phosphate, and esters (such as alkyl esters) thereof. In someembodiments, the charged substituent is sulfonate. Examples of chargedcationic substituents include quaternary amines (—NR₃ ⁺), where R isindependently selected from C₁₋₆ alkyl, aryl, and arylalkyl. Othercharged cationic substituents include protonated primary, secondary, andtertiary amines, and well as guanidinium. In some embodiments, thecharged substituent is —N(CH₃)₃ ⁺.

In some embodiments, the ionic group consists solely of a chargedsubstituent. Example charged substituents include any of those mentionedabove, including sulfonate and —N(CH₃)₃ ⁺.

In further embodiments, the ionic group corresponds to a C₁₋₂₀ alkylgroup substituted with one or more charged substituents, wherein theC₁₋₂₀ alkyl group is optionally further substituted with 1, 2, 3, 4, 5,or 6 substituents independently selected from halo, cyano, nitro, andC₁₋₄ haloalkyl, wherein 0, 1, 2, 3, 4, 5, or 6 carbon atoms of the alkylgroup are individually replaced with O, S, C(O), C(O)O, NR′, C(O)NR′,SO, SO₂, SO₂NR′, wherein R′ is H or C₁₋₆ alkyl, with the proviso thatthe replacement does not result in an unstable moiety (e.g., —O—O—,—O—S—, etc.).

Example ionic groups include groups of Formula:

wherein y and z are independently selected from 0, 1, 2, 3, 4, 5, 6, 7,and 8. In some embodiments, y and z are independently selected from 1,2, 3, 4, 5, 6, 7, and 8. In some embodiments, y and z are independentlyselected from 1, 2, 3, and 4. In some embodiments, y and z are 0.

Further example ionic groups include groups of Formula:

In some embodiments, the ionic group can contain two or more chargedsubstituents. For example, the ionic group can include both an anionicand a cationic substituent, forming a “Zwitterionic group” (or“Zwitterion”). Zwitterionic groups can be particularly useful assubstituents in the present invention because they incorporateadditional formal charges in the conjugate yet do not impact net totalcharge, thereby facilitating charge-balance. In some embodiments, aZwitterionic group corresponds to a C₁₋₂₀ alkyl group substituted withat least one positively charged (cationic) substituent and at least onenegatively charged (anionic) group, such that the overall charge of theZwitterionic group is zero, and wherein the C₁₋₂₀ alkyl group isoptionally further substituted with 1, 2, 3, 4, 5, or 6 substituentsindependently selected from halo, cyano, nitro, and C₁₋₄haloalkyl,wherein 0, 1, 2, 3, 4, 5, or 6 carbon atoms of the C₁₋₂₀ alkyl group areindividually replaced with O, S, C(O), C(O)O, NR′, C(O)NR′, SO, SO₂,SO₂NR′, wherein R′ is H or C₁₋₆ alkyl, with the proviso that thereplacement does not result in an unstable moiety (e.g., —O—O—, —O—S—,etc.).

Example Zwitterion groups comprise both a sulfonate group and aquaternary amine of formula —NR₄ ⁺, wherein R is independently selectedfrom C₁₋₆ alkyl, aryl, and arylalkyl. For example, the Zwitterionicgroup has Formula:

wherein w is 1, 2, 3, 4, 5, 6, 7, or 8.

In some embodiments, the Zwitterionic group has the Formula:

As used herein, the phrase “non-ionic oligomeric or polymericsolubilizing groups” refers to soluble polymers such as, for example,polyethylene glycol, polypropylene glycol, polyethylene oxide andpropylene oxide copolymer, a carbohydrate, a dextran, polyacrylamide,and the like. The solubilizing group can be attached by any desiredmode. The point of attachment can be, e.g., a carbon-carbon bond, acarbon-oxygen bond, or a nitrogen-carbon bond. The attachment group canbe, e.g., an ester group, a carbonate group, a ether group, a sulfidegroup, an amino group, an alkylene group, an amide group, a carbonylgroup, or a phosphate group.

Some examples of solubilizing groups include polyethylene glycols, suchas —(CH₂CH₂O)_(a)—H, —OC(═O)O(CH₂CH₂O)_(a)H, —OC(—O)O(CH₂CH₂O)_(a)CH₃,—O(CH₂CH₂O)_(a)CH₃, and —S(CH₂CH₂O)_(a)CH₃, “a” being an integer betweenabout 2 and about 250. In some embodiments, “a” is 4 to 12 or 5 to 10.In further embodiments, “a” is 6, 7, or 8. Other examples ofsolubilizing groups include dextrans such as —OC(═O)O(dextran).

The solubilizing moiety can have an absolute molecular weight of fromabout 500 amu to about 100,000 amu, e.g., from about 1,000 amu to about50,000 amu or from about 1,500 to about 25,000 amu.

In some embodiments, R⁹ and R¹⁰ are non-ionic oligomeric or polymericsolubilizing groups.

Further examples of solubilizing groups include:—(CH₂)_(c)—(OCH₂CH₂)_(d)—OR^(a), wherein “c” is 0 to 6, “d” is 1 to 200,and R^(a) is H or C₁₋₆ alkyl. In some embodiments, “c” is 1 to 4, “d” is1 to 10, and R^(a) is H. In some embodiments, “d” is 6 or 7.

See WO 2008017074, U.S. Ser. No. 12/376,243 (filed Feb. 3, 2009), andU.S. Ser. No. 12/376,225 (filed Feb. 3, 2009), each of which isincorporated herein by reference in its entirety, for a furtherdescription of suitable non-ionic oligomeric or polymeric solubilizinggroups, and method for incorporating them into dyes.

As used herein, “reactive linking group” (L′) refers to any molecularentity having a molecular weight from about 50 to about 500 Da that iscapable of conjugating with a targeting ligand (TL). In particular, thereactive linking group includes at least one reactive group selectedfrom a carboxylic acid group or anhydride or ester thereof, as well asan isothiocyanate group. In some embodiments, the reactive linking groupcontains a carboxylic acid group.

In some embodiments, L′ has the Formula:

wherein:

E is absent, O or S;

Q is (CH₂)_(q) or a non-ionic oligomeric or polymeric solubilizingmoiety;

R¹⁷, R¹⁸, R¹⁹, and R²⁰ are independently selected from H, an ionicgroup, a non-ionic oligomeric or polymeric solubilizing moiety, halo,cyano, nitro, C₁₋₄ alkyl, C₁₋₄ alkoxy, and C₁₋₄ haloalkyl;

R²¹ is H or N-succinimidyl; and

-   -   q is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

In various embodiments, all or some of the following, or any combinationof the following, may be true: E is absent or O; Q is (CH₂)_(q); Q isCH₂CH₂; R¹⁷, R¹⁸, R¹⁹, and R²⁰ are each H; R²¹ is H; R²¹ isN-succinimidyl; and q is 0.

The moiety —C(O)O—R²¹ represents the reactive moiety of the reactivelinking group that is capable of covalently attaching to a targetingligand. Accordingly, R²¹ can be H, to form the carboxy group which isreactive with amines or other nucleophiles. R²¹ can also representcarboxyl activating substituents such as N-succidimidyl (NHS) which canfacilitate conjugation.

Similarly, the “linking group” (L) is a divalent derivative of L′ in theconjugates of the invention and has the same characteristics identifiedabove except that the reactive group is covalently attached to theconjugated targeting ligand.

In some embodiments, L has the Formula:

wherein:

E is absent, O or S;

Q is (CH₂)_(q) or a non-ionic oligomeric or polymeric solubilizingmoiety;

J is C(O), C(O)O, or C(O)NH;

R¹⁷, R¹⁸, R¹⁹, and R²⁰ are independently selected from H, an ionicgroup, a non-ionic oligomeric or polymeric solubilizing moiety, halo,cyano, nitro, C₁₋₄ alkyl, C₁₋₄ alkoxy, and C₁₋₄ haloalkyl; and

-   -   q is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

In various embodiments, all or some of the following, or any combinationof the following, may be true: E is absent or O; Q is (CH₂)_(q); Q isCH₂CH₂; R¹⁷, R¹⁸, R¹⁹, and R²⁰ are each H; q is 0; and J is C(O).

In some embodiments, L has the Formula:

In some embodiments, L has the Formula:

See, Lee, H. et al. J. Org. Chem. (2006) 71(20), 7862-7865, incorporatedherein by reference in its entirety.

As used herein, the term “methine” refers to a —CH═ group. Similarly,the term “polymethinc” refers to a chain of —CH═ groups containing, forexample, 2 to 20 carbon atoms. In some embodiments, the polymethinegroup has 3 to 13 carbon atoms. In further embodiments, the polymethinegroup has 3, 5, 7, 9, 11, or 13 carbon atoms. In yet furtherembodiments, the polymethine group is a heptamethine having 7 carbonatoms.

As used herein, the term “alkyl” is meant to refer to a saturatedhydrocarbon group which is straight-chained or branched. Example alkylgroups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl andisopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl, t-butyl), pentyl(e.g., n-pentyl, isopentyl, sec-pentyl, neopentyl), and the like. Analkyl group can contain from 1 to about 20, from 2 to about 20, from 1to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 4, orfrom 1 to about 3 carbon atoms.

As used herein, “haloalkyl” refers to an alkyl group having one or morehalogen substituents. Example haloalkyl groups include CF₃, C₂F₅, CHF₂,CCl₃, CHCl₂, C₂Cl₅, and the like.

As used herein, “aryl” refers to monocyclic or polycyclic (e.g., having2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example,phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and thelike. In some embodiments, aryl groups have from 6 to about 20 carbonatoms. In some embodiments, the aryl group is phenyl.

As used herein, “cycloalkyl” refers to non-aromatic cyclic hydrocarbonoptionally including on or more unsaturations. Cycloalkyl groups caninclude mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) groupsand spirocycles. Ring-forming carbon atoms of a cycloalkyl group can beoptionally substituted by oxo or sulfido. Cycloalkyl groups also includecycloalkylidenes. Example cycloalkyl groups include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl,cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbomyl, norpinyl,norcarnyl, adamantyl, and the like. Also included in the definition ofcycloalkyl are moieties that have one or more aromatic rings fused(i.e., having a bond in common with) to the cycloalkyl ring, forexample, benzo or thienyl derivatives of cyclopentane, cyclopentene,cyclohexane, and the like. A cycloalkyl group containing a fusedaromatic ring can be attached through any ring-forming atom including aring-forming atom of the fused aromatic ring. In some embodiments, thecycloalkyl group is a 5-7 membered saturated cycloalkyl group.

As used herein, “carbocycle” or “carbocyclic” refers to an aryl orcycloalkyl group.

As used herein, “heteroaryl” refers to an aromatic heterocycle having atleast one heteroatom ring member such as sulfur, oxygen, or nitrogen.Heteroaryl groups include monocyclic and polycyclic (e.g., having 2, 3or 4 fused rings) systems. Examples of heteroaryl groups include withoutlimitation, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl,furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl,pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl,pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl,isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl,indolinyl, and the like. In some embodiments, any ring-forming N in aheteroaryl moiety can be substituted by oxo. In some embodiments, theheteroaryl group has from 1 to about 20 carbon atoms, and in furtherembodiments from about 3 to about 20 carbon atoms. In some embodiments,the heteroaryl group contains 3 to about 14, 4 to about 14, 3 to about7, or 5 to 6 ring-forming atoms. In some embodiments, the heteroarylgroup has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In someembodiments, the heteroaryl group is a 5- or 6-membered heteroaryl ring.

As used herein, “heterocycloalkyl” refers to non-aromatic heterocycleshaving one or more ring-forming heteroatoms such as an O, N, or S atom.Heterocycloalkyl groups include monocyclic and polycyclic (e.g., having2, 3 or 4 fused rings) systems as well as spirocycles. Example“heterocycloalkyl” groups include morpholino, thiomorpholino,piperazinyl, tetrahydrofuranyl, tetrahydrothienyl,2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-1,4-dioxane, piperidinyl,pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl,oxazolidinyl, thiazolidinyl, imidazolidinyl, and the like. Ring-formingcarbon atoms and heteroatoms of a heterocycloalkyl group can beoptionally substituted by oxo or sulfido. Also included in thedefinition of heterocycloalkyl are moieties that have one or morearomatic rings fused (i.e., having a bond in common with) to thenonaromatic heterocyclic ring, for example phthalimidyl, naphthalimidyl,and benzo derivatives of heterocycles. The heterocycloalkyl group can beattached through a ring-forming carbon atom or a ring-formingheteroatom. The heterocycloalkyl group containing a fused aromatic ringcan be attached through any ring-forming atom including a ring-formingatom of the fused aromatic ring. In some embodiments, theheterocycloalkyl group has from 1 to about 20 carbon atoms, and infurther embodiments from about 3 to about 20 carbon atoms. In someembodiments, the heterocycloalkyl group contains 3 to about 14, 4 toabout 14, 3 to about 7, or 5 to 6 ring-forming atoms. In someembodiments, the heterocycloalkyl group has 1 to about 4, 1 to about 3,or 1 to 2 heteroatoms. In some embodiments, the heterocycloalkyl groupcontains 0 to 3 double or triple bonds. In some embodiments, theheterocycloalkyl group contains 0 to 2 double or triple bonds. In someembodiments, the heterocycloalkyl group is a 5-, 6-, or 7-membered ring.

As used herein, “heterocycle” or “heterocyclic” refers to a heteroarylgroup or heterocycloalkyl group.

As used herein, “halo” includes fluoro, chloro, bromo, and iodo.

As used herein, “arylalkyl” refers to alkyl substituted by aryl. Anexample arylalkyl group is benzyl.

As used herein, “alkoxy” refers to an —O-alkyl group. Example alkoxygroups include methoxy, ethoxy, propoxy (e.g., n-propoxy andisopropoxy), t-butoxy, and the like. At various places in the presentspecification, substituents are disclosed in groups or in ranges. It isspecifically intended that the invention include each and everyindividual subcombination of the members of such groups and ranges. Forexample, the term “C₁₋₆ alkyl” is specifically intended to individuallydisclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl, and C₆ alkyl.

It is further appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, canalso be provided in combination in a single embodiment. Conversely,various features of the invention which are, for brevity, described inthe context of a single embodiment, can also be provided separately orin any suitable subcombination.

Compounds of the invention can also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.For example, isotopes of hydrogen include tritium and deuterium.

The chemical substances represented herein by name, chemical formula, orstructure are meant to include all stereoisomers, geometric isomers,tautomers, resonance structures, and isotopes of the same, unlessotherwise specified.

The chemical substances described herein may be charged or includesubstituents with formal charges. When such chemical substances arerepresented as charged, it is understood that, unless otherwisespecified, the charges are generally countered with an appropriatecounterion. For example, chemical substances or functional groups havinga charge of −1 are understood to be countered with an ion have a +1charge. Suitable counterions with +1 charge include Na+, K+,tetraalkylammonium ions, and the like. Conversely chemical substances orfunctional groups having a charge of +1 are understood to be counteredwith an ion having a −1 charge. Suitable counterions with −1 chargeinclude F—, Cl—, Br—, I—, perchlorate, acetate, trifluoroacetate, andthe like.

Methods of Preparing Dyes and Conjugates

The present invention further provides methods for preparing dyes andconjugates suitable for the imaging methods described herein. In someembodiments, the methods include: (a) selecting a dye having peakabsorption at about 500 nm to about 850 nm and peak fluorescent emissionat about 550 nm to about 875 nm; (b) optionally modifying the dye toinclude a linking group; and (c) modifying the dye, and optionally thelinking group, to include one or more ionic groups to achieve asolubility of the dye of at least about 10 μM in 10 mM HEPES solution atpH 7.4; wherein the one or more ionic groups are selected so that thenet charge of the dye is +1, 0, or −1, and wherein thesignal-to-background ratio of fluorescent emission detected from the dyecompound while imaging is at least about 1.1.

The present invention further provides methods of preparing a conjugatefor imaging tissue or cells, wherein the conjugate includes a dye and atargeting ligand. These methods include: (a) selecting a dye having peakabsorption at about 500 nm to about 850 nm and peak fluorescent emissionat about 550 nm to about 875 nm; (b) optionally modifying the dye toinclude a linking group; (c) modifying the dye and optionally thelinking group to include one or more ionic groups to achieve asolubility of at least about 10 μM in 10 mM HEPES solution at pH 7.4;and (d) conjugating the targeting ligand to the dye optionally throughthe linking group to form the conjugate, wherein the targeting ligandand the one or more ionic groups are selected so that the net charge ofthe conjugate is +1, 0, or −1, and wherein the signal-to-backgroundratio of fluorescent emission detected from the conjugate while imagingis at least about 1.1

The dyes described herein can be synthesized according to standardprocedures known in the art of organic chemistry. Numerous preparationof Cyanine dyes have been published. Accordingly, the dyes of theinvention can be prepared according to any of the known literaturemethods. See, for example, Mojzych, M. et al. “Synthesis of CyanineDyes” Top. Heterocycl. Chem. (2008) 14:1-9; Sysmex Journal International(1999), Vol. 9, No. 2, pg 185 (appendix); Strekowski, L. et al.Synthetic Communications (1992), 22(17), 2593-2598; Strekowski, L. etal. J. Org. Chem. (1992) 57, 4578-4580; Narayanan, N. et al. J. Org.Chem. (1995), 60(8), 2391-2395; Makin, S. M. et al. Journal of OrganicChemistry of the USSR (1977) 13(6), part 1, 1093-1096; Lee, H. et al. J.Org. Chem. (2006) 71, 7862-7865, WO 2009/006443, WO 2008/015415, WO2007/136996, WO 2007/005222, WO 2003/082988, WO 2001/090253, U.S. Ser.No. 12/376,243 (filed Feb. 3, 2009), and U.S. Ser. No. 12/376,225 (filedFeb. 3, 2009), each of which is incorporated herein by reference in itsentirety.

The dyes, conjugates, and imaging agents can be isolated as salts,acids, bases, or combinations thereof. For example, dyes, conjugates,and imaging agents having multiple charged substituents can be isolatedby introducing counterions and/or protons sufficient to counter thecharges of the various substituents normally present in neutral pH sothat the dye, conjugate, or imaging agent can be isolated, for example,as a solid substance.

Applications, Properties, and Compositions

The conjugates described herein can be used for, e.g., opticaltomographic, endoscopic, photoacoustic, and sonofluorescent applicationsfor the detection, imaging, and treatment of tumors and otherabnormalities. The conjugates can also be used for localized therapy.This can be accomplished, e.g., by attaching a porphyrin or otherphotodynamic therapy agent to a conjugate; directing the conjugates to adesired target site, or allowing the conjugates to accumulateselectively in the target site; shining light of an appropriatewavelength to activate the agent. Thus, the new conjugates can be usedto detect, image, and treat a section of tissue, e.g., a tumor.

In addition, the conjugates can be used to detect the presence of tumorsand other abnormalities by monitoring the blood clearance profile of theconjugates, for laser assisted guided surgery for the detection of smallmicrometastases of, e.g., somatostatin subtype 2 (SST-2) positivetumors, and for diagnosis of atherosclerotic plaques and blood clots.

The conjugates can be formulated into diagnostic and therapeuticcompositions for enteral or parenteral administration. Generally, thesecompositions contain an effective amount of the conjugate, along withconventional pharmaceutical carriers and excipients appropriate for thetype of administration contemplated. For example, parenteralformulations include the dye or dye conjugate in a sterile aqueoussolution or suspension. Parenteral compositions can be injected directlyinto a subject at a desired site, or mixed with a large volumeparenteral composition for systemic administration. Such solutions canalso contain pharmaceutically acceptable buffers and, optionally,electrolytes, such as sodium chloride.

Formulations for enteral administration, in general, can containliquids, which include an effective amount of the desired dye or dyeconjugate in aqueous solution or suspension. Such enteral compositionscan optionally include buffers, surfactants, and thixotropic agents.Compositions for oral administration can also contain flavoring agents,and other ingredients for enhancing their organoleptic qualities.

Generally, the diagnostic compositions are administered in doseseffective to achieve the desired signal strength to enable detection.Such doses can vary, depending upon the particular dye or dye conjugateemployed, the organs or tissues to be imaged, and the imaging equipmentbeing used. For example, Zeheer et al., Nature Biotechnology, 19,1148-1154 (2001) uses 0.1 μmol/kg as a dose for IRDye78 conjugates invivo. The diagnostic compositions can be administered to a patientsystemically or locally to the organ or tissue to be imaged, and thenthe patient is subjected to the imaging procedure.

Generally, the conjugates or dye compounds absorb and emit light in thevisible and infrared region of the electromagnetic spectrum, e.g., theycan emit green, yellow, orange, red light, or near infrared light(“NIR”).

In some embodiments, the dyes emit and/or absorb radiation having awavelength from about 300 nm to about 1000 nm, e.g., from about 400 nmto about 900 nm, or from about 450 nm to about 850 nm.

In some embodiments the conjugates and dye compounds have a maximumexcitation and/or a maximum emission, measured in 10 mM HEPES solution,pH 7.4, of from about 525 nm to about 875 nm, e.g., from about 550 nm toabout 825 nm, or from about 550 nm to about 800 nm.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention or claims in any manner. Avariety of noncritical parameters in these examples can be changed ormodified to yield essentially the same results.

EXAMPLES Example 1 Dyes

FIG. 1 depicts certain dyes that can be used in the imaging methods ofthe invention. Note that for molecules ZW−3, ZW−1, ZW+1, ZW+3, and ZW+5,a targeting ligand would have to be introduced having a +3, +1, −1, −3,and −5 charge, respectively, to achieve neutrality.

Example 2 Preparation of Dye ZW+1

Dye ZW+1 (see FIG. 1) was made according to the synthetic proceduredepicted in FIG. 2. Sodium 4-hydrazinylbenzenesulfonate (1) was reactedwith 3-methylbutan-2-one to form the 2,3,3-trimethyl-3H-indolederivative (2). The indole nitrogen was then capped by reaction with3-brorno-N,N,N-trimethylpropan-1-aminium bromide, and the product (3)reacted with the2-chloro-3-((phenylamino)methylene)cyclohex-1-enyl)methylene)benzenaminiumcompound (4) to yield the Cyanine dye (5). A linking group was attachedby reaction of (5) with 3-(4-hydroxyphenyl)propanoic acid to form theZW+1 dye (6). See also Mojzych, M. et al. “Synthesis of Cyanine Dyes”Top. Heterocycl. Chem. (2008) 14:1-9; Sysmex Journal International(1999), Vol. 9, No. 2, pg 185 (appendix); Strekowski, L. et al.Synthetic Communications (1992), 22(17), 2593-2598; Strekowski, L. etal. J. Org. Chem. (1992) 57, 4578-4580; and Makin, S. M. et al. Journalof Organic Chemistry of the USSR (1977) 13(6), part 1, 1093-1096, eachof which is incorporated herein by reference in its entirety.

Example 3 Preparation of Dye ZW+5

Dye ZW+5 (see FIG. 1) can be made according to the synthetic proceduredepicted in FIG. 3. The first step involves alkylation of indolenine 7.The resultant quaternary salt 8 can be allowed to react with3-aminopropanoyl chloride (in the foliii of an ammonium salt to protectthe amino group). Then the terminal amino group can be quatemized withexcess of methyl iodide to give the bis-quaternary salt 9. Thesubsequent steps 9→10→11 are analogous to the synthesis described inExample 2 above. Ion exchange can be used to prepare final products witha single counter anion such as chloride or bromide.

The presence of a quaternary ammonium cation close to the aromatic ringmay inhibit formation of the final dye. Accordingly, a short spacerbetween the aromatic ring and the ammonium group of ZW+5 can beintroduced.

Purification can be accomplished through silica gel chromatography orreverse-phase chromatography under both high and low-pressureconditions. Size exclusion chromatography may also be useful.

Example 4 Characterization of Dyes

After purification to homogeneity, optical properties of the dyes can bemeasured in 100% calf serum. Absorbance spectrometry (200 to 870 nm) canbe performed using a USB2000 fiber optic spectrometer andCHEM2000-UV-VIS light source with cuvette holder (Ocean Optics, Dunedin,Fla.). Fluorescence spectrometry (200 to 1100 nm) can be performed usinga HR2000 fiber optic spectrometer, CUV-ALL-UV 4-way cuvette holder(Ocean Optics), and a 250 mW 770 nm laser diode (Electro OpticalComponents, Santa Rosa, Calif.). Quantum yield can be measured underconditions of matched absorbance and 770 nm laser excitation, using ICGin DMSO (QY=13%) as the calibration standard.

Example 5 Preparation of Charge-Balanced Conjugates

Prior to in vivo testing, dyes are converted into “charge-balanced”imaging agents with net charge=0, so that they recapitulate the netcharge after conjugation to a targeting ligand. Purely anionic orcationic charge can be introduced through a free carboxylic acid on thelinking group of the dyes using, for example, amino-adamantanederivatives, described in detail previously (Maison, W., J. V.Frangioni, and N. Pannier, Synthesis of rigid multivalent scaffoldsbased on adamantane. Org Lett, 2004. 6: 4567-9; Nasr, K., N. Pannier, J.V. Frangioni, and W. Maison, Rigid Multivalent Scaffolds Based onAdantantane. J Org Chem, 2008; and U.S. Pat. App. Pub. No. 20060063834,each of which is incorporated herein by reference.

Briefly, to introduce cationic “balancing” groups, one, two or three ofthe available bridgehead carboxylic acid groups ofamino-tri-carboxy-adamantane (ATCA) (see FIG. 9) can be conjugated toeither (2-aminoethyl)trimethylammonium chloride or2,2′-iminobis(N,N,N-trimethylethanaminium chloride), and any remainingcarboxylic acids blocked with ethanolamine, to create molecules with +1,+2, +3, +4, +5, and +6 charge. Similarly, to create balancing groupswith −1, −2, −3, −4, −5, and −6 charge, the three bridgehead carboxylicacids of ATCA will be either uncapped (i.e., remaining carboxylicacids), capped with ethanolamine, or conjugated to glutamate. Theappropriate balancing group can be conjugated covalently to each dye toproduce a final conjugate, having net charge=0, prior to in vivocharacterization.

Example 6 Imaging of Organisms

The FLARE™ Image-Guided Surgery System is a continuous-wave (CW)intraoperative imaging system that is capable of simultaneous, real-timeacquisition and display of color video (i.e., surgical anatomy) and twochannels of invisible NIR fluorescent (700 nm and 800 nm) light. Detailsof the theory, engineering, and operation of the imaging system has beendescribed in detail previously. See, Tanaka, E., H. S. Choi, H. Fujii,M. G. Bawendi, and J. V. Frangioni, Image-guided oncologic surgery usinginvisible light: completed pre-clinical development for sentinel lymphnode mapping. Ann Surg Oncol, 2006. 13: 1671-81; De Grand, A. M. and J.V. Frangioni, An operational near-infrared fluorescence imaging systemprototype for large animal surgery. Technol Cancer Res Treat, 2003. 2:553-562; and Nakayama, A., F. del Monte, R. J. Hajjar, and J. V.Frangioni, Functional near-infrared fluorescence imaging for cardiacsurgery and targeted gene therapy. Molecular Imaging, 2002. 1: 365-377,each of which is incorporated herein by reference.

Specifications for the FLARE™ Image-Guided Surgery System is provided inTable 1 below.

TABLE 1 FLARE ™ NIR Fluorescence Imaging System Specifications CategorySpecification Description Physical Size Mobile Cart: 32″ W × 32″ D ×41.4″ H; Mast Height: 82″ Weight 675 lbs, including all electronics Arm6-degree-of-freedom; Reach: 43″-70″ from floor, 50.7″ from cartElectrical Voltage and Plug 120 V AC, 60 Hz; single NEMA 5-15 120 V/15 AAC plug Current 15 A max Grounding Isolation transformer for allcomponents; redundant chassis grounding Leakage Current <300 μA (perAAMI/IEC #60601) Sterility Shield Disposable acrylic shield with ≧95%transmission Drape Disposable, custom-fit plastic drape bonded to shieldLight Housing Anodized aluminum with secondary 400 W cooling plateSource Elements Custom 25 mm circular LED arrays w/integrated lineardrivers Electronics Custom passive and active boards with embeddedcontroller Fluence Rates 40,000 lx white light (400-650 nm), 4 mW/cm² of700 nm (656-678 nm) excitation light, 14 mW/cm² of 800 nm (745-779 nm)excitation light Optics Working Distance 18″ from surface of patientField-of-View 2.2 W × 1.7 H cm to 15 W × 11.3 cm (adjustable zoom)Emission/Reflectance Color Video (400-650 nm), 700 nm fluorescence (689-Channels 725 nm), 800 nm fluorescence (800-848 nm), all withsimultaneous acquisition Pixel Resolution 640 × 480 for each cameraSystem Resolution 125 × 125 μm (x, y) to 625 × 625 μm (x, y) DisplayRefresh Up to 15 Hz simultaneous acquisition on all 3 camera NIRExposure Time Adjustable from 100 μsec to 8 sec Hands-Free OpticsAutomatic zoom/focus Control 6-pedal footswitch Monitors Number 2cart-mounted 20″ for operator; 1 satellite 20″ on stand for surgeon

Example 7 In vivo Characterization of Dyes and Conjugates

For in vivo characterization, 40 pmol/g (average 10 nmol) of each dye orconjugate can be injected IV into 250 g Sprague-Dawley rats whose majorviscera are surgically exposed. The FLARE™ imaging system can be set toa 760 nm excitation fluence rate of 5 mW/cm2. Simultaneous color videoand NIR fluorescence (800 nm) images can be acquired pre-injection,every 1 sec for the first 20 sec then every 1 min for 2 h. Cameraacquisition can be held constant (typically 100 msec) and chosen toensure that all intensity measurements are within the linear range ofthe 12-bit Orca-AG (Hamamatsu) NIR camera. Blood can be sampled at 0, 1,2, 5, 10, 15, 30, 60, and 120 min via tail vein. Intensity-time curvesfor all major organs and tissues can be quantified. The peakfluorescence intensity and time can be determined for each tissue/organ,along with the intensity in each at 1 h post-injection. The experimentcan then be repeated in pigs, with measurement of NIR fluorescentintensity of skin and all internal tissues and organs. A statisticaljustification for rat and pig usage can be found in Vertebrate Animals.

Example 8 In vitro Optical and Stability Properties of Dyes

Five heptamethine indocyanine dyes, ranging in net charge from −4 to +2are shown in FIG. 4 and were characterized with respect to their opticalproperties and stability in vitro. Commercial NIR fluorophores, such asIRDye™800-RS (RS-800), IRDye800-CW (CW-800), Cy5.5, and Cy7 have variousdegrees of sulfonation in order to achieve aqueous solubility. NIRfluorophore MM-25 (+2 net charge) was prepared by employing quaternaryammonium cations (quats). MM-19 was synthesized by employing bothsulfonate groups and quats, following the synthetic scheme outlined inFIG. 2 (see Example 2). Note that MM-19 (ZW+1, see FIG. 1) has a netcharge of zero.

Table 2 below summarizes the optical and stability properties of each ofthese dyes in 100% calf serum (supplemented with 50 mM HEPES, pH 7.4).

TABLE 2 Optical Properties of Variously Charged NIR Fluorophores in 100%serum. Net Charge Extinction Stability NIR (Individual Peak CoefficientPeak at 4 h., Fluorophore MW charges) Absorbance (M⁻¹ cm⁻¹) Emission QY37° C. CW-800 1,069 −4 (−5, +1) 786 nm 237,000 801 nm 14.2% 95% RS-800865 −2 (−3, +1) 784 nm 240,000 800 nm 16.9% 97% ICG 774 −1 (−2, +1) 807nm 121,000 822 nm 9.3% 96% MM-19 1,230  0 (−3, +3) 773 nm 249,000 790 nm13.7% 95% MM-25 1,026 +2 (+3, −1) 772 nm 309,000 790 nm 16.1% 97%

Example 9 Comparative in Vivo Behavior of Dyes

While the in vitro optical and stability properties for the five testeddyes in Example 8 were similar, the in vivo behavior of these dyes wasshown to be dramatically different. Results are shown in FIG. 5. Thedyes were injected IV into rats at a dose of 40 pmol/g (10 nmol). Shownin FIG. 5 are the color video (top row) and 800 nm NIR fluorescence(bottom row) images of all major organs and tissues, surgically exposed.Excitation fluence rate was 5 mW/cm2. Camera integration time was 200msec. All NIR fluorescence images have identical normalizations.Bl=bladder. Li=Liver. In=Intestines.

As can be seen in FIG. 5, the dye with net charge of 0 (MM-19)outperformed the other dyes. The dyes with −1 (ICG) or −2 (RS-800) netcharge and having a high “hydrophobic moment” (i.e., one half ofmolecule is highly hydrophobic and the other half is hydrophilic),resulted in undesirable rapid uptake by the liver (i.e., short bloodhalf-life) and eventual excretion into bile. The dye with −4 net charge(CW-800) was cleared equally by liver and kidneys, resulting in highintestinal signal, but also demonstrated relatively high retention inskin and other major tissues and organs. The dye with +2 net charge(MM-25) was cleared by kidney more than liver, however, non-specificuptake in organs and tissue was relatively high. Finally, MM-19, whichhas a net charge of zero, demonstrated rapid equilibration betweenintravascular and extravascular spaces, no measurable liver uptake,rapid renal excretion into urine, and extremely low background retentionin normal tissues and organs.

These results were confirmed in pig (FIG. 6). All dyes were injected IVinto pigs at a dose of 40 pmol/g (1.6 μmol). Shown in FIG. 6 are thecolor video and NIR fluorescence (800 nm) images of skin, along with themeasured SBR of skin 1 h post-injection. Excitation fluence rate was 5mW/cm2. Camera integration time was 200 msec. All NIR fluorescenceimages had identical normalizations. Even after only 1 h of clearance,MM-19 signal in skin was only 11% of peak fluorescence (FIG. 6), and nonon-specific uptake was seen in any other tissues and organs. This is incontrast to ICG, RS-800, and CW-800, which resulted in very high uptakein pig liver and intestines. Blood half-lives of CW-800, RS-800, ICG,MM-19, and MM-25 were 30.6, 6.5, 4.6, 13.4, and 44.0 min, respectively.

Example 10 Comparative in Vivo Behavior of Conjugates

Dyes CW-800 (see FIG. 4) with net charge −4 and MM-19 (see FIG. 4) withnet charge of 0 were conjugated with cRGD, a specific binder to α_(v)β₃integrin. See FIG. 7 for structures of the conjugates.

Athymic nu/nu mice with integrin α_(v)β₃-expressing tumors on the leftflank (T+; arrows) and integrin α_(v)β₃-negative tumors (T-; arrowheads)on the right flank were used in this experiment. Shown are color video(top row) and 800 NIR fluorescence images (bottom row) at 0 and 4 hafter IV injection of 40 pmol/g (1 nmol) of cRGD-CW-800 (left) andcRGD-MM-19 (right)

Even though net charge on the cRGD-MM-19 conjugate was +1 due to theindole nitrogen, the results were clear that this conjugate had superiorimaging properties. After IV injection of identical doses of cRGD-MM-19and cRGD-CW-800, the MM-19 based conjugate had a much highertumor-to-background ratio (TBR) at all time points and exhibited rapidrenal clearance (see FIG. 8). The CW-800 based conjugate (net charge −3)had very high non-specific uptake in skin, muscle, and bone (FIG. 8). At4 h post-injection, cRGD-CW-800 had a TBR of 5.0 and cRGD-MM-19 had aTBR of 17.2, corresponding to a 3.4-fold improvement in TBR.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of imaging tissue or cells, the method comprising: (a)contacting the tissue or cells with an imaging agent comprising a dye orconjugate thereof, the conjugate comprising a targeting ligand attachedto the dye, wherein the dye or conjugate has a net charge of +1, 0, or−1 and comprises one or more ionic groups; (b) irradiating the tissue orcells at a wavelength absorbed by the dye or conjugate; (c) detecting anoptical signal from the irradiated tissue or cells, wherein thesignal-to-background ratio of the detected optical signal is at leastabout 1.1, thereby imaging the tissue or cells.
 2. The method of claim1, wherein; the signal-to-background ratio is at least about 1.2; thesignal-to-background ratio is at least about 1.3; thesignal-to-background ratio is at least about 1.4; the net charge is 0;the net charge is +1; the net charge is −1; the dye or conjugatecomprises two or more ionic groups; the dye or conjugate comprises threeor more ionic groups; the dye or conjugate comprises four or more ionicgroups; the imaging agent is a conjugate of the dye; the imaging agenthas a solubility in 10 mM HEPES solution, pH 7.4, of at least about 10μM; the imaging agent has peak absorbance at about 750 nm to about 825nm; the imaging agent has peak fluorescent emission at about 775 nm toabout 850 nm; the tissue or cells are abnormal or diseased; the imagingagent is administered to an organism comprising the tissue or cells; thetargeting ligand binds to selectively to prostate tissue; the targetingligand binds selectively to prostate-specific membrane antigen (PSMA);the targeting ligand binds selectively to tissue expressing α_(v)β₃integrin; or the targeting ligand comprises an RGD peptide. 3-11.(canceled)
 12. The method of claim 2, wherein the dye portion of theconjugate comprises one or more ionic groups; or wherein both the dyeand the targeting ligand portions of the conjugate each comprise one ormore ionic groups.
 13. (canceled)
 14. The method of claim 12, whereinthe total formal charge of the one or more ionic groups of the targetingligand wholly or partially cancels the total formal charge of the one ormore ionic groups of the dye. 15-19. (canceled)
 20. The method of claim2, wherein the organism is human. 21-24. (canceled)
 25. The method ofclaim 1, wherein the dye or conjugate has Formula I:

wherein: n is +1, 0, or −1; W is a C₁ methine or a C₂₋₁₃ polymethinegroup optionally substituted with 1, 2, 3, or 4 substituentsindependently selected from C₁₋₆ alkyl, a reactive linking group, or amoiety comprising a linking group and a targeting ligand, wherein twosubstituents, together with the atoms to which they are attached andoptionally one or more methine groups of W, optionally form a 5-, 6-, or7-membered carbocycle or a 5-, 6-, or 7-membered heterocycle, whereinsaid carbocycle or heterocycle is optionally substituted with 1, 2, 3,4, 5, or 6 substituents independently selected from an ionic group, anon-ionic oligomeric or polymeric solubilizing group, halo, C₁₋₆ alkyl,aryl, and heteroaryl; Ring A and Ring B are independently selected froma monocyclic heterocycle, a bicyclic heterocycle, a tricyclicheterocycle, a monocyclic a carbocycle, a bicyclic carbocycle, and atricylic carbocycle, wherein one of Rings A and B is optionally charged;R^(A) and R^(B) are independently selected from H, an ionic group, anon-ionic oligomeric or polymeric solubilizing group, halo, C₁₋₆ alkyl,aryl, and heteroaryl, wherein said alkyl, aryl, and heteroaryl groupsare optionally substituted with 1, 2, 3, 4, or 5 groups independentlyselected from halo, cyano, nitro, and C₁₋₄ haloalkyl; x and y areindependently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11;wherein at least one R^(A) and R^(B) is present and is an ionic group.26. The method of claim 25, wherein the dye or conjugate has Formula II:

wherein: n is +1, 0, or −1; W is a C₁ methine or a C₂₋₁₃ polymethinegroup optionally substituted with 1, 2, 3, or 4 substituentsindependently selected from C₁₋₆ alkyl, a reactive linking group, or amoiety comprising a linking group and a targeting ligand, wherein twosubstituents, together with the atoms to which they are attached andoptionally one or more methine groups of W, optionally form a 5-, 6-, or7-membered carbocycle or a 5-, 6-, or 7-membered heterocycle, whereinsaid carbocycle or heterocycle is optionally substituted with 1, 2, 3,4, 5, or 6 substituents independently selected from an ionic group, anon-ionic oligomeric or polymeric solubilizing group, halo, C₁₋₆ alkyl,aryl, and heteroaryl; Ring A and Ring B are independently selected frommonocyclic heterocycle, bicyclic heterocycle, and tricyclic heterocycle;R^(A) and R^(B) are independently selected from H, an ionic group, anon-ionic oligomeric or polymeric solubilizing group, halo, C₁₋₆ alkyl,aryl, and heteroaryl, wherein said alkyl, aryl, and heteroaryl groupsare optionally substituted with 1, 2, 3, 4, or 5 groups independentlyselected from halo, cyano, nitro, and C₁₋₄ haloalkyl; R^(A′) and R^(B′)are independently selected from an ionic group, a non-ionic oligomericor polymeric solubilizing group, C₁₋₆ alkyl, aryl, and heteroaryl,wherein said alkyl, aryl, and heteroaryl groups are optionallysubstituted with 1, 2, 3, 4, or 5 groups independently selected fromhalo, cyano, nitro, and C₁₋₄ haloalkyl; and u and t are independentlyselected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; wherein at least oneR^(A), R^(B), R^(A′), R^(B′), and R^(W) is present and is an ionicgroup.
 27. The method of claim 26, wherein the dye or conjugate hasFormula III:

wherein: G is independently selected from H, C₁₋₆ alkyl, a moietycomprising a linking group, and a moiety comprising a targeting ligand;Y is O, S, CR¹¹R¹²NR^(n), —CR¹¹, —CR¹¹═CR¹²—, or —CR¹¹═N—; R¹, R², R³,R⁴, R⁵, R⁶, R⁷, and R⁸ are independently selected from H, an ionicgroup, a non-ionic oligomeric or polymeric solubilizing group, halo,C₁₋₆ alkyl, aryl, and heteroaryl, wherein said alkyl, aryl, andheteroaryl groups are optionally substituted with 1, 2, 3, 4, or 5groups independently selected from halo, cyano, nitro, and C₁₋₄haloalkyl; or two adjacent R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ groups,together with the atoms to which they are attached, form a fused 5-7membered aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group, eachoptionally substituted with 1, 2, 3, 4, or 5 substituents independentlyselected from halo, cyano, nitro, and C₁₋₄ haloalkyl; R⁹, R¹⁰, and R^(n)are independently selected from an ionic group, a non-ionic oligomericor polymeric solubilizing group, C₁₋₆ alkyl, aryl, and heteroaryl,wherein said alkyl, aryl, and heteroaryl groups are optionallysubstituted with 1, 2, 3, 4, or 5 groups independently selected fromhalo, cyano, nitro, and C₁₋₄ haloalkyl; R¹¹ and R¹² are independentlyselected from C₁₋₄ alkyl optionally substituted with 1, 2, or 3 halo;R¹⁵ and R¹⁶ are independently selected from H and C₁₋₆ alkyl; or R¹⁵ andR¹⁶ together with the atoms to which they are attached and optionallyone or more —CH═ moieties, form a 6-membered aryl or cycloalkyl group,each optionally substituted ionic oligomeric or polymeric solubilizinggroup, halo, C₁₋₆ alkyl, aryl, and heteroaryl; and v is 0, 1, 2, 3, 4,or 5, wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, andR¹⁰ is an ionic group.
 28. The method of claim 27, wherein: no more thanone G is a moiety comprising a linking group or a moiety comprising atargeting ligand; or the dye or conjugate has Formula IV:

wherein R¹³ and R¹⁴ are independently selected from C₁₋₄ alkyloptionally substituted with 1, 2, or 3 halo.
 29. (canceled)
 30. Themethod of claim 28, wherein the dye or conjugate has Formula V:

wherein R²², R²³, R²⁴, R²⁵, R²⁶, and R²⁷ are independently selected fromindependently an ionic group, a non-ionic oligomeric or polymericsolubilizing group, halo, C₁₋₆ alkyl, aryl, and heteroaryl.
 31. A methodof preparing a dye for imaging tissue or cells or of preparing aconjugate for imaging tissue or cells, wherein the conjugate comprises adye and a targeting ligand, the method comprising: (1)(a) selecting adye having peak absorption at about 500 nm to about 850 nm and peakfluorescent emission at about 550 nm to about 875 nm; (b) optionallymodifying the dye to include a linking group; and (c) modifying the dye,and optionally the linking group, to include one or more ionic groups toachieve a solubility of the dye of at least about 10 μM in 10 mM HEPESsolution at pH 7.4; wherein the one or more ionic groups are selected sothat the net charge of the dye is +1, 0, or −1, and wherein thesignal-to-background ratio of fluorescent emission detected from the dyecompound while imaging is at least about 1.1; or (2)(a) selecting a dyehaving peak absorption at about 500 nm to about 850 nm and peakfluorescent emission at about 550 nm to about 875 nm; (b) optionallymodifying the dye to include a linking group; (c) modifying the dye andoptionally the linking group to include one or more ionic groups toachieve a solubility of at least about 10 μM in 10 mM HEPES solution atpH 7.4; and (d) conjugating the targeting ligand to the dye optionallythrough the linking group to form the conjugate, wherein the targetingligand and the one or more ionic groups are selected so that the netcharge of the conjugate is +1, 0, or −1, and wherein thesignal-to-background ratio of fluorescent emission detected from theconjugate while imaging is at least about 1.1.
 32. (canceled)
 33. A dyeor an imaging agent, wherein: the imaging agent is for imaging tissue orcells, wherein the imaging agent comprises a conjugate which ischaracterized as having detectable fluorescence with asignal-to-background ratio of at least about 1.1, and wherein theconjugate has Formula VI:

wherein: TL is a targeting ligand comprising at least one binding moietythat binds to a biological target; L is a linking group; R¹, R², R³, R⁴,R⁵, R⁶, R⁷, and R⁸ are independently selected from H, an ionic group, anon-ionic oligomeric or polymeric solubilizing group, halo, C₁₋₆ alkyl,aryl, and heteroaryl, wherein said alkyl, aryl, and heteroaryl groupsare optionally substituted with 1, 2, 3, 4, or 5 groups independentlyselected from halo, cyano, nitro, and C₁₋₄ haloalkyl; or two adjacentR¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ groups, together with the atoms towhich they are attached, form a fused 5-7 membered aryl, heteroaryl,cycloalkyl, or heterocycloalkyl group, each optionally substituted with1, 2, 3, 4, or 5 substituents independently selected from halo, cyano,nitro, and C₁₋₄ haloalkyl; R⁹ and R¹⁰ are independently selected from anionic group, a non-ionic oligomeric or polymeric solubilizing group,C₁₋₆ alkyl, aryl, and heteroaryl, wherein said alkyl, aryl, andheteroaryl groups are optionally substituted with 1, 2, 3, 4, or 5groups independently selected from halo, cyano, nitro, and C₁₋₄haloalkyl; R₁₁, R₁₂, R₁₃, and R¹⁴ are independently selected from C₁₋₄alkyl optionally substituted with 1, 2, or 3 halo; R¹⁵ and R¹⁶ areindependently selected from H and C₁₋₆ alkyl; or R¹⁵ and R¹⁶ togetherwith the —CH═CH—CH═ moiety which they span form a 6-membered aryl orcycloalkyl group, each optionally substituted with 1, 2, 3, or 4substituents independently selected from an ionic group, a non-ionicoligomeric or polymeric solubilizing group, halo, C₁₋₆ alkyl, aryl, andheteroaryl; n is 1, 0, or −1; wherein at least one of R¹, R², R³, R⁴,R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ is an ionic group; or the dye comprises amolecule or ion of Formula VIII:

wherein: L′ is a reactive linking group; R¹, R², R³, R⁴, R⁵, R⁶, R⁷, andR⁸ are independently selected from H, an ionic group, a non-ionicoligomeric or polymeric solubilizing group, halo, C₁₋₆ alkyl, aryl, andheteroaryl, wherein said alkyl, aryl, and heteroaryl groups areoptionally substituted with 1, 2, 3, 4, or 5 groups independentlyselected from halo, cyano, nitro, and C₁₋₄ haloalkyl; or two adjacentR¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ groups, together with the atoms towhich they are attached, form a fused 5-7 membered aryl, heteroaryl,cycloalkyl, or heterocyclocalkyl group, each optionally substituted with1, 2, 3, 4, or 5 substituents independently selected from halo, cyano,nitro, and C₁₋₄ haloalkyl; R⁹ and R¹⁰ are independently selected from anionic group, a non-ionic oligomeric or polymeric solubilizing group,C₁₋₆ alkyl, aryl, and heteroaryl, wherein said alkyl, aryl, andheteroaryl groups are optionally substituted with 1, 2, 3, 4, or 5groups independently selected from halo, cyano, nitro, and C₁₋₄haloalkyl; R¹¹, R¹², R¹³, and R¹⁴ are independently selected from C14alkyl optionally substituted with 1, 2, or 3 halo; R¹⁵ and R¹⁶ areindependently selected from H and C₁₋₆ alkyl; or R¹⁵ and R¹⁶ togetherwith the —C═C—C— moiety which they span form a 6-membered aryl orcycloalkyl group, each optionally substituted with 1, 2, 3, or 4substituents independently selected from an ionic group, a non-ionicoligomeric or polymeric solubilizing group, halo, C₁₋₆, alkyl, aryl, andheteroaryl; and p is −6, −5, −4, −3, −2, −1, 0, +1, +2, +3, +4, +5, or+6; wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰is an ionic group comprising at least one cationic substituent; or thedye has Formula X:


34. The imaging agent of claim 33, wherein: n is 0; n is +1; n is −1; atleast two of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are ionicgroups; at least three of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰are ionic groups; at least four of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,and R¹⁰ are ionic groups; or the ionic group is independently selectedfrom: (a) a charged substituent; and (b) a C₁₋₂₀ alkyl group substitutedwith one or more charged substituents, and optionally substituted with1, 2, 3, 4, 5, or 6 substituents independently selected from halo,cyano, nitro, and C₁₋₄ haloalkyl, wherein 0, 1, 2, 3, 4, 5, or 6 carbonatoms of the alkyl group are individually replaced with O, S, C(O),C(O)O, NR′, C(O)NR′, SO, SO₂, SO₂NR′, wherein R′ is H or C₁₋₆ alkyl,with the proviso that the replacement does not result in an unstablemoiety. 35-40. (canceled)
 41. The imaging agent of claim 34, wherein:the charged substituent is selected from sulfonate or a quaternary amineof formula —NR₄ ⁺, wherein R is independently selected from C₁₋₆ alkyl,aryl, and arylalkyl; the C₁₋₂₀ alkyl group substituted with one or morecharged substituents has the Formula:

wherein y and z are independently selected from 1, 2, 3, 4, 5, 6, 7, and8; the C₁₋₂₀ alkyl group substituted with one or more chargedsubstituents is independently selected from:

the C₁₋₂₀ alkyl group substituted with one or more charged substituentsis a Zwitterionic group; R¹¹ R¹², R¹³, and R¹⁴ are each methyl; R¹⁵ andR¹⁶ together with the —C═C—C— moiety which they span form a 6-memberedaryl or cycloalkyl group, each optionally substituted with 1, 2, 3, or 4substituents independently selected from an ionic group, a non-ionicoligomeric or polymeric solubilizing group, halo, C₁₋₆ alkyl, aryl, andheteroaryl; R¹⁵ and R¹⁶ together form —CH₂—CH₂—CH₂—; L has the formula:

wherein: E is absent, O or S; Q is (CH₂)_(q) or a non-ionic oligomericor polymeric solubilizing moiety; J is C(O), C(O)O, or C(O)NH; R¹⁷, R¹⁸,R¹⁹, and R²⁰ are independently selected from H, an ionic group, anon-ionic oligomeric or polymeric solubilizing moiety, halo, cyano,nitro, C₁₋₄ alkyl, C₁₋₄ alkoxy, and C₁₋₄ haloalkyl; and q is 0, 1, 2, 3,4, 5, 6, 7, or 8; or the conjugate has Formula VII:

wherein at least one of R², R⁶, R⁹, and R¹⁰ is an ionic group. 42-44.(canceled)
 45. The imaging agent of claim 41, wherein the Zwitterionicgroup comprises a sulfonate group and a quaternary amine of formula —NR₄⁺, wherein R is independently selected from C₁₋₆ alkyl, aryl, andarylalkyl. 46-50. (canceled)
 51. The imaging agent of claim 41, wherein:n is +1; two of R², R⁶, R⁹, and R¹⁰ are cationic groups; two of R², R⁶,R⁹, and R¹⁰ are cationic groups and two of R², R⁶, R⁹, and R¹⁰ areanionic groups; or the conjugate has the Formula VIII:

wherein: n is 0, +1, or −1; and TL is a targeting ligand. 52-55.(canceled)
 56. The dye compound of claim 33: wherein L′ comprises a—COOH group or a —C(O)O—NHS group; wherein L′ has the formula:

wherein: E is absent, O or S; Q is (CH₂)_(q) or a non-ionic oligomericor polymeric solubilizing moiety; R¹⁷, R¹⁸, R¹⁹, and R²⁰ areindependently selected from H, an ionic group, a non-ionic oligomeric orpolymeric solubilizing moiety, halo, cyano, nitro, C₁₋₄ alkyl, C₁₋₄alkoxy, and C₁₋₄ haloalkyl; R²¹ is H or N-succinimidyl; and q is 0, 1,2, 3, 4, 5, 6, 7, or 8; or comprising a molecule or ion of Formula IX:

wherein at least one of R², R⁶, R⁹, and R¹⁰ is an ionic group comprisingat least one cationic substituent. 57-58. (canceled)
 59. The dye ofclaim 56, wherein p is
 0. 60. The dye of claim 59, wherein at least twoof R², R⁶, R⁹, and R¹⁰ are ionic groups each comprising at least onecationic substituent.
 61. (canceled)
 62. A conjugate comprising the dyeof claim 33 conjugated to a targeting ligand, wherein the total netcharge of said conjugate is −1, 0, or +1.
 63. An imaging agentcomprising the conjugate of claim
 62. 64. The dye of claim 33, isolatedas a salt, acid, or combination thereof.